1. Field of the Invention
The present invention relates to the use for inhibition of tumor cell induced platelet aggregation of admixtures of at least two members of: (1) calcium channel blocker compounds, known for use in the treatment of cardiovascular disorders, such as hypertension, angina and arrhythmia; (2) thromboxane synthase inhibitors, known for use in antithrombotic therapy to inhibit platelet aggregation; (3) phosphodiesterase inhibitors, known for their ability to inhibit platelet aggregation; and (4) prostacyclin (and prostacyclin analogs and stimulating agents), known for their ability to inhibit platelet aggregation. In particular, the present invention preferably relates to the use of admixtures of nimodipine (Bay e 9736 nifedipine (Bay a 1040), diltiazem and all other structurally related and unrelated calcium channel blocking compounds in combination with U63557A UK 38485 CGS 14854 and all other thromboxane synthase inhibitors; RX-RA 69 and all other phosphodiesterase inhibitors, or prostacyclin, prostacyclin analogs and prostacyclin
2. Prior Art
The primary focus of cancer therapy and research has been directed towards the treatment of the initial or primary tumor. Considerable success has been achieved by utilizing surgery, radiotherapy, and/or chemotherapy. However, it has become increasingly apparent that metastasis, the spread of cancer from the initial tumor to other physically separate sites, is an equally life-threatening situation that must be confronted. The exact steps of metastasis are not known, but it has been demonstrated, that the entrapment or adhesion of circulating tumor cells to endothelial substrata is an important step of the metastatic cascade.
Briefly, the metastatic cascade can be described as a sequence of events that a tumor cell or cells must successfully complete in order to become a metastatic foci. Although different texts vary somewhat in their terminology, the cascade can be thought of as four sequential stages or steps. First, a tumor cell or clump of tumor cells must be "shed" by the primary tumor. Second, the tumor cells must enter the vascular or lymphatic system and avoid destruction by host immune defenses (macrophages, natural killer cells, immune complexes, etc.). Third, the tumor cells must adhere to the endothelial lining or subendothelial lining of the vascular or lymphatic system. Fourth, the adhering tumor cells must avoid dislodgement, extravasate through the endothelium and divide. Tumor cell interactions with host platelets to form a tumor cell-platelet aggregate or thrombus has been shown to be a mechanism that allows tumor cells to successfully complete the last stages of the metastatic cascade.
Platelet aggregation and adhesion are typically thought to be initiated by a number of soluble and non-soluble factors including catecholamines, arachidonic acid metabolites (prostaglandin E2, thromboxane A2), immune complexes, complement components, ADP, and collagen (Gordon, J. L. In: Platelets: Pathophysiology and Antiplatelet Drug Therapy. Alan R. Liss, Inc., New York, pp. 13-17, 1982; Weiss, H. J. In: Platelets: Pathophysiology and Antiplatelet drug Therapy. Alan R. Liss, Inc., New York pp. 13-17, 1982; and Jamieson, G. A., et al. In: Interaction of Platelets and Tumor Cells. Alan R. Liss, Inc., New York, pp. 405-413). Additionally, it has been demonstrated that tumor cells induce platelet aggregation (Gasic, G. J., et al., Int. J. Cancer 11:704-718, 1973; Hara, H., et al., Cancer Res. 40:1217-1222, 1980; and Bastida, E., et al., Nature 291:661-662, 1981). The resultant tumor cell-platelet thrombus can protect the tumor cells from attack by the host immune system, increase the likelihood that the tumor cells would adhere to the endothelial lining or subendothelial lining of the vascular system, and protect adhering tumor cells from dislodgement. Thus, pharmacological agents that inhibit platelet aggregation or reduce platelet number have been investigated for their ability to suppress metastasis (Gasic, G. J., et al., Proc. Natl. Acad. Sci. USA 61:46-52, 1968; Honn, K. V., et al., Science 212:1270-1272, 1981; and Menter, D., et al., In: Prostaglandins and Cancer. First Intl. Conf., pp. 809-813, 1982). Calcium channel blockers are described in U.S. patent application Ser. No. 480,704 (WSU 4.1-2) for this purpose.
Platelet response to aggregating stimuli is a rapid and usually consists of a shape change from the normal discoid shape to a rounding-up, followed by the extrusion of long, thin pseudopodia. Mobilization of Ca.sup.++ from intracellular stores (probably the dense tubular system) may be the trigger for the shape change and apparently precedes the primary platelet aggregation and the associated dense, alpha, and lysosomal granule release (Shaw, J. O. et al. Biochim. Biophys. Acta 714:492-499, 1982). Although the exact mechanism has not been elucidated, we believe that experimental evidence suggests that an influx of extracellular calcium is required for secondary platelet aggregation (Imai, A., et al., Biochem. Biophys. Res. Commun. 108:(2)752-759, 1982). Both primary and secondary platelet aggregation are associated with phospholipase activation, arachidonic acid metabolism and the formation of cyclooxygenase and lipoxygenase products by stimulated platelets. Phospholipase A2 and C are activated by the increase in intraplatelet Ca.sup.++ concentration from "resting" levels of 10.sup.-8 to 10.sup.-7 M to activated levels of 10.sup.-5 to 10.sup.-3 M (Gerrard, J. M., et al., In: Platelets in Biology and Pathology--2. J. L. Gordon, ed., North Holland Biomedical Press, Amsterdam, pp. 407-436, 1981). The two phospholipases, however, may be triggered sequentially and not simultaneously by increases in intraplatelet Ca.sup.++. Phospholipase C may be activated in the primary phase of platelet aggregation by the release of internal stores of Ca.sup.++ (Rittenhouse-Simmons, S. J. Biol. Chem. 256:(9)4153-4155, 1981; Shukla, S. D. Life Sciences 30:1323-1335, 1982). The activated phospholipase C degrades phosphatidyl inositol which in turn is phosphorylated to phosphatidic acid. Phosphatidic acid may serve as an ionophore to trigger an influx of extra cellular calcium which may activate phospholipase A2 (Serhan, C. N, et al., J. Biol. Chem. 257:(9) 4746-4752, 1982). This activation results in an additional increase phosphatidic acid, the eventual formation of thromboxane A2 (a cyclooxygenase product) and secondary aggregation (Gorman, R. R. Fed. Proc. 38:(1)83-88, 1979; Parise, L. V., et al., J. Pharm. Expotl. Therap. 222:(1)276-281, 1982). Compounds derived from arachidonic acid (prostacyclin, PGI.sub.2 and thromboxane A.sub.2, TXA.sub.2) have been demonstrated to have a profound but possibly not exclusive (Vargaftig, B. B., et al Biochem Pharmacol. 30 263-271 (1981)) role in platelet aggregation and normal hemostasis. The prior art has demonstrated the formation of TXA.sub.2 from the endoperoxide intermediate PGH2 (Hamberg, M., et al., Proc. Natl. Acad. Sci. USA 72:2994, 1975). Subsequently platelet TXA.sub.2 biosynthesis was found to be stimulated by numerous aggregating agents and was believed to be an absolute requirement for platelet aggregation (Gorman, R. R., Fed. Proc. 38:83, 1979). This view has recently been challenged by the observations that in some cases the endoperoxide PGH.sub.2 can initiate platelet aggregation independent of its conversion to TXA.sub.2 (Heptinstall, S., et al., Thromb. Res. 20:219, 1980).
One year following the discovery of TXA.sub.2, Vane and co-workers (Moncada, S., et al., Nature 263:663, 1976) discovered PGI.sub.2 as a transformation product of prostaglandin endoperoxides. Prostacyclin is produced by vascular tissue of all species so far tested and is the main product of arachidonic acid metabolism in isolated vascular tissue. Prostacyclin is the most potent endogenous inhibitor of platelet aggregation yet discovered, being 30 to 40 times more potent than PGE.sub.1 (Moncada, S. et al., In: Biochemical Aspects of Prostaglandins and Thromboxanes, (Eds. N. Kharasch and J. Fried), P. 155. Academic Press, New York, 1977) and 1000 times more potent than adenosine (Mullane, K. M., et al., Eur. J. Pharmacol. 54:217, 1979). In addition, PGI.sub.2 can reverse secondary platelet aggregation in vitro (Moncada, S., et al., Prostaglandins. 12:715, 1971) and in the circulatory system of man (Szczeklik, A., et al., J. Pharmacol. Res. Commun. 10:545, 1978). It has been suggested that PGI.sub.2 and TXA.sub.2 play an antagonistic and pivotal role in the control of thrombosis centered upon their bidirectional (PGI.sub.2 increases, TXA.sub.2 decreases) effect on platelet cAMP levels.
It has been demonstrated that tumor metastasis is enhanced by tumor cell interactions with platelets and that agents which block or prevent tumor cell-platelet interaction and aggregation have antimetastatic effects (Gastpar, H., J. Medicine 8(2):103-114, 1977; Bastida, E., et al., Cancer Res. 42:4348-4352, 1982; Paschen, W., et al., P. Blut 38:17-24, 1979; Honn, K. V., et al., Acta Clinica Belgica, in press, 1983; and Honn, K. V., et al., Biochem. Pharm., in press, 1983). Agents which have been investigated, function by reducing platelet cell number in the blood or by inhibiting platelet function (aggregation). Recently, calcium channel blockers (verapamil and nifedipine) have been reported to inhibit platelet aggregation induced by epinephrine or ADP (Owen, N. E., et al., Am. J. Physiol. 239:483-488, 1980; Schumunk, G. A. et al., Res. Commun. Chem. Path. and Pharm. 35:(2)179-187, 1982 and Owen, N. E. et al., Am. J. Physiol. 241:613-619, 1981). Because of these recent reports of the inhibitory effects of calcium channel blockers upon platelet aggregation and investigations concerning the interactions between platelet antiaggregatory agents and metastasis, the antimetastatic effects of BAY e 9736 (nimodipine) have been investigated by use in a number of different in vivo and in vitro assay systems as described in U.S. application Ser. No. 480,704. We have demonstrated that the chronic administration of nimodipine to mice will significantly reduce spontaneous metastasis and that prior treatment of nimodipine will reduce metastasis induced by tail vein injection of B16a tumor cells to syngeneic mice. In vitro, we have demonstrated that nimodipine will greatly inhibit platelet aggregation induced by tumor cells and ADP, and increase the reversal rate of aggregated platelets induced by ADP or tumor cells. We have also demonstrated that nimodipine decreases the rate of tumor cell growth over a five day assay period, decreases the rate of incorporation of .sup.3 H-thymidine into tumor cell DNA, and decreases or inhibits the adhesion of tumor cells to plastic incubation flasks and a virally transformed endothelial cell monolayer.
Our working hypothesis has been that the normal intravascular balance between prostacyclin (PGI.sub.2) and platelet arachidonic acid metabolities (i.e., TXA.sub.2) can be altered by the presence of a primary tumor and/or circulating tumor cells and their shed (membrane) vesicles. This hypothesis predicts that arachidonic acid metabolism by the tumor cell, platelet and vessel wall is a fundamental determinant in the sum total of their interaction and that PGI.sub.2 may be a natural deterrent to metastasis. We have demonstrated that PGI.sub.2 is a potent inhibitor of tumor cell induced platelet aggregation (TCIPA) in vitro, and platelet enhanced adhesion of tumor cells to plastic, and endothelial cells (Menter, et al., Cancer Research, 44, 450-456, 1984). We have also demonstrated that PGI.sub.2 is a potent antimetastatic agent in vivo and have evidence that endogenous PGI.sub.2 production may limit metastasis (Honn K., et al Science 212 1270-1272 (1981). We have also postulated that thromboxane synthase inhibitors could also serve as antimetastatic agents, due to their inhibition of TXA.sub.2 induced platelet aggregation in response to tumor cells. In vitro the TX synthase inhibitors of the endoperoxide class inhibit tumor cell induced platelet aggregation (TCIPA) and in vivo inhibit lung colonization from tail vein injected tumor cells. Honn, K., et al J. of Clinical and Exp. Metastasis 1 103-114 (1983). However, imidazole type TX synthase inhibitors do not inhibit TCIPA even in the presence of inhibited TXA.sub.2 production. However, these compounds possess antimetastatic activity in vivo in both the tail vein ("experimental metastasis") and spontaneous metastasis models. To explain these apparent discrepancies it is proposed that the in vivo antimetastatic activity of TX synthase inhibitors is due to operation of the "steal hypothesis" which suggests that an inhibition of platelet TXA.sub.2 biosynthesis would cause the release of PGH.sub.2, the substrate of both prostacylin and thromboxane synthases, which would be absorbed by the vascular endothelium and/or leukocytes. This would cause enhanced PGI.sub.2 biosynthesis by the endothelium of the vascular wall and/or by leukocytes.